Abstract: Wheat gluten as raw materials which was the byproducts of wheat starch, effected of enzymolysis time, substrate concentration, enzyme concentration, pH and temperature on the hydrolysis degree of wheat gluten through single factor experiment and orthogonal experiment, we explored the optimum process conditions of enzymaticing hydrolysis of wheat gluten. Studies found that the order of five factors effect on the degree of hydrolysis of wheat gluten from strong to weak was:pH, enzymolysis temperature, enzyme concentration, substrate concentration, enzymolysis time. The results showed that wheat gluten had the highest degree of hydrolysis when reaction time was 3h, 9% of the substrate concentration, enzyme concentration was 4%, pH8.0, enzymolysis temperature was 55℃, when the degree of hydrolysis of the wheat gluten was 22.96%. In the optimal hydrolysis conditions, we also measured the molecular weight of gluten polypeptides through Tricine SDS-PAGE, which was in the range of 8.04~50.12 ku.